<p>There is a prevalent belief that acute hormone responses to resistance exercise mediate adaptations in skeletal muscle hypertrophy; however, there is little supporting evidence. We conducted studies to examine the relationship between acute hormonal increases after resistance exercises and subsequent changes in muscle anabolism.</p> <p>We tested the hypothesis that exercise-induced responses of anabolic hormones—growth hormone (GH) and testosterone—would enhance rates of myofibrillar protein synthesis (MPS) after an acute bout of resistance exercise, and would augment muscle hypertrophy after training. We concluded, however, that resistance exercise-induced increases in putative anabolic hormones do not enhance MPS or hypertrophy.</p> <p>We also examined whether rates of MPS would be attenuated in women (compared with men) after resistance exercise, due to their lack of post-exercise testosteronemia. We reported similar increases in MPS in men and women; post-exercise testosterone responses in women, which were 45-fold lower than men, did not attenuate elevations in MPS.</p> <p>Collectively, our work leads to the conclusion that the acute rise in hormones such as testosterone and GH has very little bearing on MPS and hypertrophy responses to resistance exercise. Instead, the rise in these hormones appears to be a non-specific response to exercise stress rather than a response that is important for muscle anabolism. Contrary to widely used principles, our data suggests that exercise programs should not be designed based on nuances in the post-exercise hormonal milieu. Alternatively, understanding local mechanotransduction, which is directly linked to muscle fibre loading, will reveal the processes that drive human exercise-mediated muscle hypertrophy.</p> / Doctor of Philosophy (PhD)

<p>Introduction: Mitochondrial dysfunction and oxidative stress increase with aging and may contribute to age-associated muscle atrophy (sarcopenia). Resistance exercise (RE) can promote the accretion of muscle mass, increase strength, and ultimately improve function in the elderly. Such beneficial effects are thought to be mitigated solely by increased muscle mass and strength; however, the contribution of the mitochondria to the beneficial effects of RE in aging have not been thoroughly characterized. While mitochondrial benefits have been established separately in both young and aged adults following chronic RE, the acute effects have not been well characterized. Methods: Sedentary young and aged adult males completed either an acute bout of fatiguing RE or endurance exercise (EE), and muscle biopsies were obtained at 3, 24 and 48 h post- exercise depending on the study. Results: Despite equivalent lean-body mass, increased age was associated with elevated mtDNA deletions, indicating potential for mitochondrial dysfunction. RE was associated with reduced mitochondrial content (transcripts, protein, and mtDNA copy number) at 48 h post-exercise, a response that did not differ with increasing age. Paradoxically, reduced mitochondrial content occurred alongside elevated total peroxisome proliferator-activated receptor γ coactivator one α (PGC-1α) mRNA; however, RE altered only the PGC-1α4 isoform post-exercise, a transcript that regulates myostatin and insulin-like growth factor one (IGF1) signalling and ultimately muscle hypertrophy and not mitochondrial adaptations. In addition, PGC-1α modulates the unfolded protein response (UPR), and RE was subsequently shown to elevate endoplasmic reticulum stress and elicit the UPR. Conclusion: PGC-1α mRNA increases regardless of exercise mode; however, differential expression or regulation of alternate PGC-1α isoforms or transcriptional binding partners co-activated by PGC-1α may dictate the specific phenotypic adaptations that occur following divergent modes of exercise. Furthermore, RE acutely decreases mitochondrial content despite elevated PGC-1α mRNA, and this response is not influenced by age.</p> / Doctor of Philosophy (Medical Science)

<p>Energy restriction-induced weight loss results in the loss of both fat mass (FM) and fat free mass (FFM). Declines in FFM, comprised mostly of skeletal muscle, have the potential to impair metabolic health. A recent meta-analysis demonstrated higher protein hypo-energetic diets (≥ 1.25 g/kg/d) can help retain FFM (Weinheimer, Sands, & Campbell, 2010). This effect is attributed to protein ingestion induced stimulation of muscle protein synthesis (MPS), which is dependent on protein source (Tang, Moore, Kujbida, Tarnopolsky, & Phillips, 2009). However, protein source on modulating FFM preservation during ER remains understudied. In a double blind investigation, 40 adults (BMI 28-40) undertook a moderate 14 day hypo-energetic diet. Subjects were randomized to be supplemented twice daily with whey (WHEY) or soy (SOY) protein, or a carbohydrate (CHO) powder as part of their ER diet. Preliminary and post diet intervention testing included skeletal muscle biopsies, blood samples and DXA measures. Absolute changes in body mass (-2.3 ± 0.18 kg), FM (-1.2 ± 0.16 kg) and FFM (-0.8 ± 0.24 kg) did not significantly differ between groups. Basal serum cortisol was unchanged in WHEY but increased significantly in SOY and CHO (p</p> / Master of Science in Kinesiology

<p>This thesis investigated the acute and chronic responses of human skeletal muscle to high-intensity exercise, with a particular focus on markers of mitochondrial content, and the potential for nutrition to manipulate the adaptive response in recreationally active individuals. The acute response was primarily assessed via measurement of signalling proteins and mRNA species linked to exercise-induced mitochondrial biogenesis. The chronic response was determined via changes in the protein content or maximal activities of mitochondrial enzymes after training. Study 1 examined whether the manner in which a given amount of high-intensity cycling work was performed (i.e., in an intermittent or continuous fashion) altered the acute metabolic response to exercise, and whether the acute response was indicative of longer-term adaptations. Despite the similar acute activation of signalling proteins after the intermittent and continuous matched-work exercise protocols, 6 wk of training with the continuous protocol did not increase mitochondrial content, contrary to what we have previously shown after 6 wk training with the intermittent protocol. This suggests that the intermittent application of a low-volume, high-intensity stimulus is important to elicit training-induced increases in mitochondrial content. Furthermore, Study 1 showed that acute changes in specific signalling proteins did not necessarily predict chronic adaptations. Studies 2 and 3 examined whether specific nutritional interventions, previously shown to modulate acute exercise capacity or metabolic response, altered the mitochondrial adaptive response to several weeks of HIT. Neither manipulating carbohydrate availability between twice daily training sessions, or chronic ingestion of β-alanine, augmented skeletal muscle adaptations in response to 2-6 wk of HIT. It is possible that small influences of nutrition were overwhelmed by the potency of HIT, which stimulated marked increases in mitochondrial content in this population. Overall this thesis advances our basic understanding of the skeletal muscle adaptive response to HIT and the influence of nutrition.</p> / Doctor of Philosophy (PhD)

<p>This thesis examined the effect of high-intensity "sprint" interval training (SIT) on aerobic-based exercise performance and metabolic adaptations in human skeletal muscle. It has long been recognized that several weeks of interval-based training increased skeletal muscle oxidative capacity; however, little was known regarding the minimum "dose" of SIT necessary to elicit this adaptive response or the time-course and magnitude of adaptation in other markers of skeletal muscle metabolic control. Our general hypothesis was that low-volume SIT would induce rapid improvements in a wide array of metabolic variables that were comparable to traditional high-volume endurance training (ET). Healthy young men and women were recruited to perform four to six 30- second "all out" Wingate Tests, three times per week with one to two days of recovery, for up to six weeks. The weekly dose of SIT corresponded to ~10 minutes of maximal cycling exercise (-225-300 kJ) over a total training time commitment of 60-90 minutes, including recovery. The SIT response was compared against control subjects who performed no training or an ET group who performed up to one hour per day of moderate-intensity cycling exercise, five days per week for six weeks (-2250 kJ per week). Our major findings were that one to two weeks of SIT increased performance during aerobic-based exercise (time-to-fatigue tests and time-trials of varying duration) and the maximal activity or total protein content of mitochondrial enzymes and transport proteins associated with carbohydrate metabolism (e.g., citrate synthase, cytochrome oxidase, glucose transporter 4). Six weeks of SIT or ET induced similar increases in markers of skeletal muscle carbohydrate (pyruvate dehydrogenase E1a protein content) and lipid oxidation (3-hydroxyacyl CoA dehydrogenase maximal activity) and peroxisome-proliferator-activated receptorgamma coactivator-1a protein content, and similar reductions in phosphocreatine and glycogen utilization during matched-work exercise. These data suggest that SIT is a time-efficient strategy to increase skeletal muscle oxidative capacity and to induce specific metabolic adaptations during exercise that are comparable to ET.</p> / Doctor of Philosophy (PhD)

Skeletal muscle fascia is a connective tissue which provides structure and aidswith force transfer in a muscle. Currently there are no good ways of detectingand analyzing micrometer thick structures of this tissue in-vivo. In this thesis,we created a model to detect skeletal muscle fascia, and tested its performanceusing simulated data. Utilizing the ultrasound simulation software Vantage,which operates through MATLAB, we created a simulation model which repli-cates the properties and behaviour of skeletal muscle fascia. To detect thetissue, we changed and adapted a previously implemented model of ultrasoundlocalization microscopy (ULM), previously only used to create super resolutionimages of blood vessels. Finally we evaluated the models ability to locate anddetermine the thickness of the simulated fascia. Additionally we tested themodels ability to separate adjacent objects.We found that our model was successful at detecting and localizing thesimulated fascia, with a sub wavelength accuracy. The precision of the locatedfascia appears more accurate for horizontally aligned objects compared to thevertically aligned ones. The results from determining the thickness of the fasciaproved relatively successful as well. However the results showed a high variance.This could be improved through an inclusion of stocasticity in the simulationmodel we developed. Finally the ability to distinguish two objects close to eachother showed successful results as well. The method was able to clearly detecta fascia circle with a 0.5mm diameter. It was unable to detect the sides a fasciacircle with a 0.25mm diameter.The main limitation with the model we have developed lies in the simulationsperformed. The simulation model we used was very basic, meaning that it didnot perfectly represent the skeletal muscle fascia we sought to examine. Furtherdevelopment of the simulation model is required to provide a result which ismore representative of real skeletal muscle fascia.The analysis of this first model shows promise in detecting the simplifiedfascia provided by our simulation model. At this stage, the method will requiremore extensive testing, together with a more thorough statistical analysis, beforewe can state the usefulness of the method.

Branched-chain amino acids (BCAA) are a group of essential amino acids consisting of leucine, isoleucine, and valine. Leucine, in particular, has signaling functions affecting protein and energy metabolism. Plasma leucine concentration is positively correlated with obesity and associated metabolic disorders. We set out to test the hypothesis that metabolic dysfunction from high fat diets precedes dysfunctional BCAA metabolism. First, BCAA were supplemented to neonatal pigs for 4 weeks to evaluate whether the anabolic signaling function of leucine could increase muscle growth when fed for a longer duration than in previous studies. Neither normal pigs nor low birth weight pigs, which have naturally impaired muscle growth, grew better in response to BCAA supplementation, despite low birth weight pigs expressing less of the leucine sensing protein Sestrin2 in skeletal muscle. Furthermore, high plasma BCAA concentrations caused by the experimental diets had no effect on adiposity, liver fat accumulation, or expression of genes related to fatty acid synthesis, mitochondrial biogenesis, or energy expenditure in the pigs' livers. Having produced strong evidence that long term BCAA supplementation neither improves lean growth nor causes abnormal fat metabolism, we then tested whether fat supplementation changes BCAA metabolism. Pigs were fed milk replacer formula with either low energy (Control), or high energy from long-chain fatty acids (LCFA) or medium-chain fatty acids (MCFA) for 22 days. Although high fat diets did not increase plasma BCAA concentrations, the MCFA diet in particular caused metabolic changes which could lead to fatty liver disease and decreased oxidative BCAA disposal. Expression of fatty acid synthesizing genes were increased in the livers of pigs fed MCFA formula compared to Control and LCFA formula. Oxidation of α-ketoisocaproic acid was decreased in liver homogenate of pigs fed MCFA and LCFA formulas compared to Control. Additionally, hepatic oxidation of α-ketoisovalerate was decreased, and plasma concentration of α-ketoisovalerate was consequently increased, in pigs fed MCFA formula compared to Control, with LCFA formula causing intermediate results. In future research, it would be valuable to feed high MCFA formula for a longer period of time to determine whether nonalcoholic fatty liver disease will develop, and whether plasma BCAA concentrations will increase due to decreased oxidation. Overall, these studies concluded that long term BCAA supplementation does not increase muscle growth in neonatal pigs, but there is also no indication that they cause obesity or dysfunctional fat metabolism. On the other hand, high fat diets cause impairments in BCAA catabolism which may precede elevated plasma BCAA concentrations. / Doctor of Philosophy / Branched-chain amino acids (BCAA) are essential amino acids which are abundant in plant and animal proteins. In addition, the BCAA leucine has functions in protein and energy metabolism. Leucine consumption induces a signal to build new muscle protein. However, leucine concentration is also higher in blood plasma of obese individuals than in non-obese individuals, which has caused uncertainty regarding the safety of leucine consumption. In order to demonstrate that leucine does not cause obesity, we set out to test the hypothesis that high fat diets cause decreased breakdown of BCAA. In the first study, we tested whether one month of BCAA supplementation could increase muscle growth in neonatal pigs. Neither normal pigs nor low birth weight pigs, which have naturally impaired muscle growth, grew better in response to BCAA supplementation, despite low birth weight pigs expressing less of a leucine sensing protein in skeletal muscle. Furthermore, BCAA supplementation caused higher BCAA concentrations in blood plasma, but did not cause pigs to gain more fat, or cause any changes in liver fat metabolism. Having produced strong evidence that BCAA supplementation neither improves lean growth nor causes abnormal fat metabolism, we then tested whether fat supplementation changes BCAA metabolism. Pigs were fed milk replacer formula which was either low calorie (Control), or high calorie from animal fat, which is rich in long-chain fatty acids (LCFA) or high calorie from coconut oil, which is rich in medium-chain fatty acids (MCFA). Although high fat diets did not increase blood plasma BCAA concentrations, the MCFA formula in particular caused changes which could lead to fatty liver disease and decreased breakdown of BCAA. Genes which synthesize new fatty acids were increased in the livers of pigs fed MCFA formula compared to those fed LCFA and Control formulas. Furthermore, liver samples taken from pigs fed the MCFA and LCFA formulas were less able to fully break down metabolites of leucine compared to pigs fed the Control formula. In addition, liver samples from MCFA fed pigs were less able to fully break down metabolites of the BCAA valine, which led to higher concentrations of that metabolite in the blood plasma of pigs fed MCFA formula compared to pigs fed LCFA or Control formula. In the future, it would be valuable to feed a high MCFA formula for a longer period of time to determine whether nonalcoholic fatty liver disease will develop, and whether blood plasma BCAA concentrations will increase due to decreased breakdown. Overall, these studies concluded that long term BCAA supplementation does not increase muscle growth in neonatal pigs, but there is also no indication that they cause obesity or dysfunctional fat metabolism. On the other hand, high fat diets cause impairments in BCAA breakdown which may lead to elevated BCAA concentrations in blood plasma.

Growing evidence suggests that obesity and associated metabolic dysregulation occurs in concert with chronic low-grade inflammation. Toll-like receptors (TLR) are transmembrane receptors that play an important role in innate immunity and the induction of inflammatory responses. Our laboratory has observed that TLR4 expression is elevated in the skeletal muscle of obese humans and is associated with reduced fatty acid (FA) oxidation and increased lipid synthesis. Additionally, activation of this pathway by lipopolysaccharide (LPS), ex vivo, results in a shift in substrate metabolism favoring glucose as an energy substrate and preferential storage of FA in intracellular lipid depots. The purpose of this study was to examine the effects of saturated vs. monounsaturated FA on TLR4 transcription and signaling using ex vivo and in vivo models. C2C12 myotubes were incubated in FA-enriched growth medium with varying ratios of palmitate and oleate for 12 hours. Following FA treatment, cells were either collected for measures of mRNA and protein levels of TLR4 or challenged with LPS (500 ng/mL) for 2 hours to assess TLR4 mediated changes in interleukin-6 (IL-6) and glucose and fatty acid metabolism. TLR4 mRNA and protein content were increased in stepwise fashion with higher palmitate concentration (p<0.05). This was associated with an exacerbated LPS effect on IL-6 mRNA and protein levels, and glucose and fatty acid metabolism. To determine if these effects are translated to an in-vivo model, C57BL/6 mice were fed high saturated fat (HSF), high monounsaturated fat (HMF), and control diets for 10 weeks. Following the dietary intervention, animals were challenged with I.P. injections of either saline or LPS (~25μg/mouse), sacrificed 4 hours post-injection, and red and white gastrocnemius muscle were harvested for measures of expression and protein levels of TLR4 and IL-6, and glucose and fatty acid metabolism. TLR4 mRNA and protein levels were not altered with either the HSF or HMF diets. However, there was a heightened LPS response with regards to increases in IL-6 and TNF-α, and enhanced shifts in substrate metabolism following the HSF diet (p<0.05). These effects were not observed in response to the HMF diet. In conclusion, these data demonstrate that a milieu of high saturated fatty acids results in elevated sensitization of the TLR4 pathway in skeletal muscle. These results provide insight into how a westernized diet, one enriched in saturated fat, may link chronic inflammation with obesity-associated metabolic abnormalities. / Ph. D.

A promising approach to treat patients with vocal fold paralysis using electrical stimulation is investigated throughout this research work. Functional Electrical Stimulation works by stimulating the atrophied muscle or group of muscles directly by current when the transmission lines between the central nervous system are disrupted. This technique helps maintain muscle mass and promote blood flow in the absence of a functioning nervous system. The goal of this work is two-fold: develop control techniques for muscle contraction to optimize muscle stimulation and develop a small-scale electromagnetic system to provide stimulation to the laryngeal muscles for patients with vocal fold paralysis. These studies; therefore, focus on assessing a linear Proportional-Integral (PI) controller and two nonlinear controllers: Model Reference Adaptive Controller (MRAC) and an Adaptive Augmented PI (ADP-PI) system to identify the most appropriate controller providing effective stimulation of the muscle. Direct stimulation is applied to mouse skeletal muscle in vitro to test the controllers along with numerical simulations for validation of these experimental tests. The experiments included muscle contractions following four distinct trajectories: a step, sine, ramp, and square wave. Overall, the closed-loop controllers followed the stimulation trajectories set for all the simulated and tested muscles. When comparing the experimental outcomes of each controller, we concluded that the ADP-PI algorithm provided the best closed-loop performance for speed of convergence and disturbance rejection. Next, the focus of the research work was on the implementation of an electromagnetic system to generate appropriate currents of stimulation using the aforementioned controllers. For this study, Nickel-Titanium shape memory alloys were used to assess activation (contraction) through a two-coil system guided by the controllers. The application of the two-coil system demonstrated the effectiveness of the approach and a main effect was observed between the PI, MRAC, and ADP-PI controllers when following the trajectories. Lastly, a small scale two-coil system is developed for animal testing in the muscle-mass-spring setup. Experiments were successful in generating the appropriate stimulation controlled by the output-based algorithms for muscle contraction. Trials conducted for this study were compared to the muscle contractions observed in the first study. The controllers were able to provide appropriate stimulation to the muscle system to follow the set trajectories: a step, ramp, and sinusoidal input. More trials are required to draw statistical conclusions about the performance of each controller. Regardless, the small-scale two-coil system along with the applied controllers can be reconfigured to be an implantable system and tested for appropriate stimulation of the laryngeal muscles. / Ph. D.

Skeletal muscle is a primary contributor to body mass and whole-body energy metabolism. It is an adaptive tissue with the ability to fluctuate in size and mechanical properties in response to stimulus. Health conditions involving chronic elevated inflammation levels often feature metabolic inflexibility and losses in skeletal muscle mass. Mononuclear stem cells, termed satellite cells, are mitotic and serve to donate nuclei to muscle fibers to enable skeletal muscle adaptation. Despite the well-characterized nature of satellite cell activation, proliferation, and differentiation, the underlying mechanisms regulating this process is not fully understood. Recent characterization of cytokines secreted by skeletal muscle in an endocrine type fashion has led to discoveries of inflammatory cytokines influencing satellite cell function. However, how the autocrine production and secretion of these cytokines during proliferation and differentiation in humans and their correlation with myogenic transcription factors is not well understood. Our study used satellite cells cultured from the vastus lateralis of 12 male human research subjects, and ELISA analysis to measure levels of TNF-α and IL-6 across proliferation, early differentiation, and late differentiation. Additionally, mRNA levels of Pax7, MyoD, myogenin, IL-6, TNF-α, and TGF-β were assessed in satellite cells cultured from a subset of two endurance trained and two sedentary individuals from the larger group of 12 human subjects. The novelty of our study is the large number of human research subjects and simultaneous analysis of inflammatory cytokine secretion, mRNA inflammatory cytokine expression, and myogenic transcription factor mRNA expression. Results showed an 83% decrease in IL-6 protein secretion 24 hours after exposure to differentiation media (p-value <0.05) before increasing 50-fold after 7 day of exposure to differentiation media (p-value < 0.05). Myogenin and TGF-β mRNA expression levels were positively correlated (R2 = 0.5814, p-value < 0.0001). A negative correlation was found between IL-6 and MyoD (R2 = 0.2473, p – value = 0.0257). After 1 day of exposure to differentiation media, satellite cells from endurance trained subjects exhibited higher levels of TGF-β mRNA expression compared to sedentary satellite cells of sedentary subjects of the same age and levels of adiposity (p-value < 0.05). Results support a potential relationship in humans satellite cells between myogenic transcription factors and inflammatory cytokines, however, further study is necessary in order to investigate the underlying mechanisms behind the correlations. / Master of Science / Skeletal muscle is responsible for conscious, voluntary movement. In addition, the tissue is responsible for the majority of energy expenditure in the human body. Skeletal muscle is able to adapt to exercise programs through the fusion of undifferentiated stem cells – called satellite cells – in the skeletal muscle fiber. In long-term diseased conditions, the immune response involves chronic rises in inflammation and results in the loss of skeletal muscle and corresponding loss of ability to move. A shorter rise in inflammation is also linked with the positive exercise response. Our study features satellite cells harvested from muscle samples of 12 male human research participants. We were interested in evaluating the relationships between the expression and secretion of two proteins associated with inflammation and regulation of the satellite cell cycle. The two proteins of interest in our study are tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). We also measure the gene expression of another inflammatory protein, transforming growth factor beta (TGF-β). In order to know where the cells were in their life cycle, we measured expression of genes associated with the division (Pax7), early fusion (MyoD), and late fusion of satellite cells (myogenin). Our study found a decrease in IL-6 secretion and expression as the process of satellite cells turning into muscle fibers was initiated. Additionally, a 50-fold increase in IL-6 expression was found at day 7 compared to day 0 of the satellite cell cycle. Additionally, we found a positive correlation between TGF-β and myogenin and a negative correlation between IL-6 and MyoD. Although we found correlations between satellite cell cycle genes and inflammation genes, more research is necessary to see if there is a pathway causing this relationship.